Expression control elements from the Lemnaceae family

ABSTRACT

Compositions and methods for regulating expression of nucleotide sequences of interest in a plant are provided. Compositions include novel nucleic acid molecules, and variants and fragments thereof, for expression control elements isolated from the  Lemnaceae  ubiquitin, r-histone and chitinase genes. A method for expressing a nucleotide sequence of interest in a plant using the expression control elements disclosed herein is further provided. The method includes introducing into a plant or plant cell or nodule an expression construct comprising an expression control element of the present invention operably linked to a nucleotide sequence of interest. In particular, the compositions and methods find use in enhancing expression of nucleotide sequences of interest in duckweed. Also provided is a novel  Lemnaceae  signal peptide-encoding sequence and the signal peptide encoded thereby. Where an expression construct of the invention is designed to express a polypeptide of interest, this novel signal peptide-encoding sequence can be included within the expression construct of the invention to provide for extracellular secretion of the encoded polypeptide of interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional patent application of U.S. patentapplication Ser. No. 11/653,593, filed Jan. 16, 2007, now U.S. Pat. No.7,622,573, which claims the benefit of U.S. Provisional PatentApplication Nos. 60/759,308, filed Jan. 17, 2006, and 60/848,961, filedOct. 3, 2006, each of which is hereby incorporated in its entirety byreference herein.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted concurrently withthe specification as a text file via EFS-Web, in compliance with theAmerican Standard Code for Information Interchange (ASCII), with a filename of 380989SequenceListing.txt, a creation date of Oct. 19, 2009, anda size of 26.7 KB. The sequence listing filed via EFS-Web is part of thespecification and is hereby incorporated in its entirety by referenceherein.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for enhancinggene expression in plants.

BACKGROUND OF THE INVENTION

The duckweeds are the sole members of the monocotyledonous familyLemnaceae. The five genera and 38 species are all small, free-floating,fresh-water plants whose geographical range spans the entire globe(Landolt (1986) Biosystematic Investigation on the Family of Duckweeds:The Family of Lemnaceae—A Monograph Study (Geobatanischen Institut ETH,Stiftung Rubel, Zurich)). Although the most morphologically reducedplants known, most duckweed species have all the tissues and organs ofmuch larger plants, including roots, stems, flowers, seeds and fronds.Duckweed species have been studied extensively and a substantialliterature exists detailing their ecology, systematics, life-cycle,metabolism, disease and pest susceptibility, their reproductive biology,genetic structure, and cell biology (Hillman (1961) Bot. Review 27:221;Landolt (1986) Biosystematic Investigation on the Family of Duckweeds:The Family of Lemnaceae—A Monograph Study (Geobatanischen Institut ETH,Stiftung Rubel, Zurich)).

The growth habit of the duckweeds is ideal for microbial culturingmethods. The plant rapidly proliferates through vegetative budding ofnew fronds, in a macroscopic manner analogous to asexual propagation inyeast. This proliferation occurs by vegetative budding from meristematiccells. The meristematic region is small and is found on the ventralsurface of the frond. Meristematic cells lie in two pockets, one on eachside of the frond midvein. The small midvein region is also the sitefrom which the root originates and the stem arises that connects eachfrond to its mother frond. The meristematic pocket is protected by atissue flap. Fronds bud alternately from these pockets. Doubling timesvary by species and are as short as 20-24 hours (Landolt (1957) Ber.Schweiz. Bot. Ges. 67:271; Chang et al. (1977) Bull. Inst. Chem. Acad.Sin. 24:19; Datko and Mudd (1970) Plant Physiol. 65:16; Venkataraman etal. (1970) Z. Pflanzenphysiol. 62: 316).

Intensive culture of duckweed results in the highest rates of biomassaccumulation per unit time (Landolt and Kandeler (1987) The Family ofLemnaceae—A Monographic Study Vol. 2: Phytochemistry, Physiology,Application, Bibliography (Veroffentlichungen des GeobotanischenInstitutes ETH, Stiftung Rubel, Zurich)), with dry weight accumulationranging from 6-15% of fresh weight (Tillberg et al. (1979) Physiol.Plant. 46:5; Landolt (1957) Ber. Schweiz. Bot. Ges. 67:271; Stomp,unpublished data). Protein content of a number of duckweed species grownunder varying conditions has been reported to range from 15-45% dryweight (Chang et al. (1977) Bull. Inst. Chem. Acad. Sin. 24:19; Changand Chui (1978) Z. Pflanzenphysiol. 89:91; Porath et al. (1979) AquaticBotany 7:272; Appenroth et al. (1982) Biochem. Physiol. Pflanz.177:251). Using these values, the level of protein production per literof medium in duckweed is on the same order of magnitude as yeast geneexpression systems.

Duckweed plant or duckweed nodule cultures can be efficientlytransformed with an expression cassette containing a nucleotide sequenceof interest by any one of a number of methods includingAgrobacterium-mediated gene transfer, ballistic bombardment, orelectroporation. Stable duckweed transformants can be isolated bytransforming the duckweed cells with both the nucleotide sequence ofinterest and a gene that confers resistance to a selection agent,followed by culturing the transformed cells in a medium containing theselection agent. See U.S. Pat. No. 6,040,498 to Stomp et al.

A duckweed gene expression system provides the pivotal technology thatwould be useful for a number of research and commercial applications.For plant molecular biology research as a whole, a differentiated plantsystem that can be manipulated with the laboratory convenience of yeastprovides a very fast system in which to analyze the developmental andphysiological roles of isolated genes. For commercial production ofvaluable polypeptides, a duckweed-based system has a number ofadvantages over existing microbial or cell culture systems. Plantsdemonstrate post-translational processing that is similar to mammaliancells, overcoming one major problem associated with the microbial cellproduction of biologically active mammalian polypeptides, and it hasbeen shown by others that plant systems have the ability to assemblemulti-subunit proteins, an ability often lacking in microbial systems(Hiatt (1990) Nature 334:469). Scale-up of duckweed biomass to levelsnecessary for commercial production of recombinant proteins is fasterand more cost efficient than similar scale-up of mammalian cells, andunlike other suggested plant production systems, for example, soybeansand tobacco, duckweed can be grown in fully contained and controlledbiomass production vessels, making the system's integration intoexisting protein production industrial infrastructure far easier.

Accordingly, there remains a need for optimized compositions and methodsfor expressing proteins of interest in duckweed.

BRIEF SUMMARY OF THE INVENTION

Compositions and methods for regulating gene expression in a plant areprovided. Compositions include novel nucleotide sequences for expressioncontrol elements (e.g., promoters and introns) isolated from Lemnaceaeubiquitin, replacement (r)-histone and chitinase genes. The expressioncontrol elements of the invention initiate transcription of operablylinked heterologous nucleotide sequences in plants. More particularly,the compositions of the invention include the expression controlelements set forth in SEQ ID NOs:1-3, 13 and 14, and variants andfragments thereof. Compositions also include novel intron sequenceswithin these Lemnaceae expression control elements, particularly theintron sequences set forth in SEQ ID NOs:7-9 and variants and fragmentsthereof. These intron sequences can be operably linked to a promoter ofinterest to enhance expression of an operably linked heterologousnucleotide sequence in a plant.

Also provided is a novel Lemnaceae chitinase signal peptide set forth inSEQ ID NO:16, encoded by a sequence set forth in SEQ ID NO:15, andvariants and fragments thereof. The signal peptide-encoding sequence canbe operably linked to a coding sequence for a polypeptide of interest todirect extracellular secretion of the encoded polypeptide.

Expression constructs (e.g., cassettes and vectors) comprising anexpression control element and/or intron and/or signal peptide-encodingsequence of the invention operably linked to a heterologous nucleotidesequence of interest are provided. Stably transformed plants, plantcells and nodules having an expression construct of the invention arefurther provided.

The compositions of the invention find use in methods directed toexpressing nucleotide sequences of interest in a plant or plant cell ornodule. The methods of the invention include introducing into a plant orplant cell or nodule an expression construct having a Lemnaceaeubiquitin, r-histone or chitinase expression control element (e.g., asset forth in SEQ ID NOs:1-3, 13 and 14), or a variant or fragmentthereof, operably linked to a nucleotide sequence of interest. Themethods of the invention further comprise introducing into a plant orplant cell or nodule an expression construct including an expressioncontrol element isolated from the Lemna gibba ribulose-1,5-bisphosphatecarboxylase small subunit gene (RbcS; e.g., as set forth in SEQ IDNOs:10-12). In other embodiments, methods of the invention includeintroducing into a plant or plant cell or nodule an expression constructhaving a Lemnaceae chitinase signal peptide-encoding sequence (e.g., asset forth in SEQ ID NO:15), or a variant or fragment thereof, operablylinked to the coding sequence for a polypeptide of interest.

In some embodiments, the methods of the invention are directed to theproduction of a polypeptide encoded by a nucleotide sequence of interestin a plant expression system (e.g., a duckweed expression system). Theplant expression system of the present invention is optimized to producehigh levels of the polypeptide sequence of interest. Thus, the inventionencompasses methods for the expression of a nucleotide sequence ofinterest in plants that are transformed with expression constructs forthe expression of the nucleotide sequence of interest, where thesenucleotide sequences are modified to enhance their expression in plants.

These and other aspects of the invention are disclosed in more detail inthe description of the invention given below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods directed tonovel nucleic acids for plant expression control elements that regulatetranscription of heterologous nucleotide sequences in plants.Specifically, the compositions of the invention comprise expressioncontrol elements isolated from the Lemnaceae ubiquitin, r-histone andchitinase genes, including the expression control elements set forth inSEQ ID NOs:1-3, 13 and 14, and variants and fragments thereof, asdefined herein below. The individual promoter (SEQ ID NOs:4-6, 13 and14) and intron (SEQ ID NOs:7-9) sequences within these expressioncontrol elements also find use in regulating transcription in plants.The invention also provides a novel L. minor chitinase signal peptide(SEQ ID NO:16) and the corresponding coding sequence (SEQ ID NO:15), andvariants and fragments thereof.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues (e.g., peptide nucleic acids) having the essential nature ofnatural nucleotides in that they hybridize to single-stranded nucleicacids in a manner similar to naturally occurring nucleotides.

The invention encompasses isolated or substantially purified nucleicacid compositions. An “isolated” or “purified” nucleic acid molecule issubstantially or essentially free from components that normallyaccompany or interact with the nucleic acid molecule or protein as foundin its naturally occurring environment. Thus, an isolated or purifiednucleic acid molecule is substantially free of other cellular material,or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. Preferably, an “isolated” nucleic acid is freeof sequences (preferably protein encoding sequences) that naturallyflank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends ofthe nucleic acid) in the genomic DNA of the organism from which thenucleic acid is derived. For example, in various embodiments, theisolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturallyflank the nucleic acid molecule in genomic DNA of the cell from whichthe nucleic acid is derived.

The compositions of the invention include isolated nucleic acidmolecules comprising the expression control element nucleotide sequencesset forth in SEQ ID NOs:1-3, 13 and 14, and variants and fragmentsthereof, as defined herein below. By “expression control element” isintended a regulatory region of DNA usually comprising a TATA boxcapable of directing RNA polymerase II to initiate RNA synthesis at theappropriate transcription initiation site for a particular codingsequence. An expression control element may additionally comprise otherrecognition sequences generally positioned upstream or 5′ to the TATAbox, which influence (e.g., enhance) the transcription initiation rate.Furthermore, an expression control element may additionally comprisesequences generally positioned downstream or 3′ to the TATA box, whichinfluence (e.g., enhance) the transcription initiation rate.

It is recognized that having identified the nucleotide sequences for theexpression control element regions disclosed herein, it is within thestate of the art to isolate and identify further regulatory elements inthe 5′ untranslated region (UTR) upstream from the particular expressioncontrol element regions identified herein. Thus, for example, theexpression control element regions disclosed herein may further compriseadditional regulatory elements such as those responsible for tissue andtemporal expression of the coding sequence, enhancers, and the like. Seeparticularly Australian Patent No. AU-A-77751/94 and U.S. Pat. Nos.5,466,785 and 5,635,618 (both of which are herein incorporated byreference).

The expression control elements of the invention were isolated fromubiquitin, r-histone and chitinase genes for several members of theLemnaceae family, and are thus referred to as “Lemnaceae expressioncontrol elements.” SEQ ID NO:1 sets forth the full-length Lemna minorubiquitin expression control element, including both the promoter plus5′ UTR (nucleotides 1-1625) and intron (nucleotides 1626-2160). SEQ IDNO:2 sets forth the full-length Spirodella polyrrhiza ubiquitinexpression control element, including both the promoter plus 5′ UTR(nucleotides 1-1041) and intron (nucleotides 1042-2021). SEQ ID NO:3sets forth the full-length Lemna aequinoctialis ubiquitin expressioncontrol element, including both the promoter plus 5′ UTR (nucleotides1-964) and intron (nucleotides 965-2068). SEQ ID NO:4 sets forth thepromoter plus 5′ UTR portion of the L. minor ubiquitin expressioncontrol element. SEQ ID NO:5 sets forth the promoter plus 5′ UTR portionof the S. polyrrhiza ubiquitin expression control element. SEQ ID NO:6sets forth the promoter plus 5′UTR portion of the L. aequinoctialisubiquitin expression control element. SEQ ID NO:7 sets forth the intronportion of the L. minor ubiquitin expression control element. SEQ IDNO:8 sets forth the intron portion of the S. polyrrhiza ubiquitinexpression control element. SEQ ID NO:9 sets forth the intron portion ofthe L. aequinoctialis ubiquitin expression control element.

SEQ ID NO:13 sets forth the full-length Lemna minor r-histone expressioncontrol element, including the promoter plus 5′ UTR. SEQ ID NO:14 setsforth the full-length Lemna minor chitinase expression control element,including the promoter plus 5′ UTR. SEQ ID NO:15 sets forth the L. minorchitinase signal peptide-encoding sequence. SEQ ID NO:16 sets forth theL. minor chitinase signal peptide.

It is recognized that the individual promoter plus 5′ UTR sequences setforth in SEQ ID NOs:4-6, 13 and 14, and biologically active variants andfragments thereof, can be used to regulate transcription of operablylinked nucleotide sequences of interest in plants. Similarly, one ormore of the intron sequences set forth in SEQ ID NOs:7-9, andbiologically active fragments or variants thereof, can be operablylinked to a promoter of interest, including a promoter set forth in SEQID NO:4, 5, 6, 13, or 14 in order to enhance expression of a nucleotidesequence that is operably linked to that promoter.

Fragments and variants of the disclosed expression control elements,signal peptide-encoding sequence, and encoded signal peptide are alsoencompassed by the present invention. By “fragment” in the context of anexpression control element is intended a portion of the full-lengthexpression control element, such as a portion of any one of theexpression control elements set forth in SEQ ID NOs:1-3, 13 and 14.Fragments of an expression control element retain biological activityand hence encompass fragments capable of initiating or enhancingexpression of an operably linked nucleotide sequence. Thus, for example,less than the entire expression control elements disclosed herein may beutilized to drive expression of an operably linked nucleotide sequenceof interest, such as a nucleotide sequence encoding a heterologousprotein. Specific, non-limiting examples of such fragments of anexpression control element include: (i) the nucleotide sequences setforth in any one of SEQ ID NOs:4-9 (as described herein above); (ii) 5′truncations of the L. minor ubiquitin expression control element (SEQ IDNO:1), such as nucleotides 1288-2160 of SEQ ID NO:1 (LmUbq truncatedpromoter No. 1, as found in the Egs22 construct described herein below)and nucleotides 1132-2160 of SEQ ID NO:1 (LmUbq truncated promoter No.2, as found in the Egs23 construct described herein below); (iii) 5′truncations of the L. minor r-histone expression control element (SEQ IDNO:13), such as nucleotides 461-1808 of SEQ ID NO:13 (LmHIS (461-1808),as found in the Egs19 construct described herein below) and nucleotides805-1808 of SEQ ID NO:13 (LmHIS (805-1808), as found in the Egs20construct described herein below); and (iv) 5′ truncations of the L.minor chitinase expression control element (SEQ ID NO:14), such asnucleotides 51-1338 of SEQ ID NO:14 (LmCHT (51-1338), as found in theEgs24 and Egs25 constructs described herein below).

As used herein, “full-length sequence” in reference to a specifiednucleotide sequence means having the entire nucleic acid sequence of anative sequence. By “native sequence” is intended an endogenoussequence, that is, a non-engineered sequence found in an organism'sgenome.

Thus, a fragment of a Lemnaceae expression control element can functionas a biologically active portion of the expression control element. Abiologically active portion of a expression control element can beprepared by isolating a portion of one of the expression controlelements of the invention and assessing the activity (e.g., the abilityto initiate or enhance transcription) of that portion of the expressioncontrol element. Nucleic acid molecules that are fragments of anexpression control element comprise at least 15, 20, 25, 30, 35, 40, 45,50, 75, 100, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800,900, 1200, 1500, 1800, or 2000 contiguous nucleotides, or up to thenumber of nucleotides present in the full-length expression controlelements disclosed herein (i.e., 2160 nucleotides for SEQ ID NO: 1, 2021nucleotides for SEQ ID NO:2, 2068 nucleotides for SEQ ID NO:3, 1808nucleotides for SEQ ID NO:13, and 1338 nucleotides for SEQ ID NO:14).

The nucleotides of such fragments will usually comprise the TATArecognition sequence of the particular expression control element. Suchfragments can be obtained by use of restriction enzymes to cleave thenaturally occurring expression control elements disclosed herein; bysynthesizing a nucleotide sequence from the naturally occurring sequenceof the expression control element DNA sequence; or can be obtainedthrough the use of polymerase chain reaction (PCR) technology. Seeparticularly, Mullis et al. (1987) Methods Enzymol. 155:335-350, andErlich, ed. (1989) PCR Technology (Stockton Press, New York). Variantsof these expression control element fragments, such as those resultingfrom site-directed mutagenesis, are also encompassed by the compositionsof the present invention.

“Fragment” in the context of a signal peptide-encoding sequence andencoded signal peptide is intended to mean a portion of the codingsequence or a portion of the signal peptide encoded thereby. Withrespect to coding sequences, fragments of a nucleotide sequence canencode polypeptide fragments that retain the biological activity of thenative polypeptide, in this case, the native L. minor chitinase signalpeptide. Thus, a functional fragment of the L. minor chitinase signalpeptide directs movement of a mature protein of interest through thesecretory pathway of a plant cell. Fragments of a coding nucleotidesequence can range from at least about 20 nucleotides, about 25nucleotides, about 50 nucleotides, about 75 nucleotides, and up to theentire nucleotide sequence encoding the L. minor chitinase signalpeptide (i.e., up to 84 nucleotides of SEQ ID NO:15).

By “variants” is intended sequences having substantial similarity withan expression control element disclosed herein (e.g., SEQ ID NOs:1-3, 13and 14) or a fragment thereof (e.g., the sequences set forth in SEQ IDNOs:4-9), or with a signal peptide-encoding sequence (e.g., SEQ IDNO:15) or a signal peptide (e.g., SEQ ID NO:16) or a fragment thereof.For nucleotide sequences, naturally occurring variants such as these canbe identified with the use of well-known molecular biology techniques,as, for example, with PCR and hybridization techniques as outlinedbelow. Variant nucleotide sequences also include synthetically derivednucleotide sequences, such as those generated, for example, by usingsite-directed mutagenesis. Generally, variants of a particularnucleotide sequence of the invention, including variants of any of SEQID NOs:1-9 and 13-15, will have at least 40%, 50%, 60%, 65%, 70%,generally at least 75%, 80%, 85%, preferably about 90%, 91%, 92%, 93%,94%, to 95%, 96%, 97%, and more preferably about 98%, 99% or moresequence identity to that particular nucleotide sequence as determinedby sequence alignment programs described herein below using defaultparameters. Biologically active variants are also encompassed by thepresent invention. Biologically active variants include, for example,the native expression control elements, or native signalpeptide-encoding sequence, of the invention having one or morenucleotide substitutions, deletions, or insertions.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid sequences or two polypeptide sequences makes reference tothe residues in the two sequences that are the same when aligned formaximum correspondence over a specified comparison window. By“comparison window” is intended a contiguous and specified segment of apolynucleotide/polypeptide sequence, where thepolynucleotide/polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. Generally, the comparison window is atleast 20 contiguous nucleotides/amino acids in length, and optionallycan be 30, 40, 50, 100 nucleotides/amino acids, or longer.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters.

The CLUSTAL program is well described by Higgins et al. (1988) Gene73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al.(1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. TheALIGN program is based on the algorithm of Myers and Miller (1988)CABIOS 4:11-17. A PAM120 weight residue table, a gap length penalty of12, and a gap penalty of 4 can be used with the ALIGN program whencomparing amino acid sequences. The BLAST programs of Altschul et al(1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin andAltschul (1990) Proc. Natl. Acad. Sci. USA 87:2264. BLAST nucleotidesearches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotidesequence of the invention. BLAST protein searches can be performed withthe BLASTX program, score=50, wordlength=3, to obtain amino acidsequences homologous to a protein or polypeptide of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST2.0) can be utilized as described in Altschul et al. (1997) NucleicAcids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be usedto perform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) Nucleic Acids Res. 25:3389. Whenutilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of therespective programs (e.g., BLASTN for nucleotide sequences and BLASTXfor proteins) can be used.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1992) Proc. Natl. Acad.Sci. USA 89: 10915). Alignment may also be performed manually byinspection.

An alternative indication that two nucleic acid molecules are closelyrelated is that the two molecules hybridize to each other understringent conditions. Stringent conditions are sequence-dependent andare different under different environmental parameters. Generally,stringent conditions are selected to be about 5° C. to 20° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Conditions for nucleic acid hybridization andcalculation of stringencies can be found, for example, in Sambrook etal. (2001) Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.) and Tijssen (1993)Hybridization With Nucleic Acid Probes, Part I: Theory and Nucleic AcidPreparation (Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Ltd., NY, N.Y.).

For purposes of the present invention, “stringent conditions” encompassconditions under which hybridization will only occur if there is lessthan 25% mismatch between the hybridization molecule and the targetsequence. “Stringent conditions” may be broken down into particularlevels of stringency for more precise definition. Thus, as used herein,“moderate stringency” conditions are those under which molecules withmore than 25% sequence mismatch will not hybridize; conditions of“medium stringency” are those under which molecules with more than 15%mismatch will not hybridize, and conditions of “high stringency” arethose under which sequences with more than 10% mismatch will nothybridize. Conditions of “very high stringency” are those under whichsequences with more than 6% mismatch will not hybridize.

Expression control element activity for any of the Lemnaceae expressioncontrol elements, or fragments or variants thereof, can be assayed usinga variety of techniques well known to one of ordinary skill in the art,including, for example, Northern blot analysis, reporter activitymeasurements taken from transcriptional fusions, and the like. See, forexample, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).Alternatively, expression control element assays may be based on themeasurement of levels of a reporter gene such as β-glucuronidase (GUS),green fluorescent protein (GFP), or the like produced under the controlof an expression control element, or fragment or variant thereof. See,for example, U.S. Pat. No. 6,072,050, herein incorporated by reference.Activity of the L. minor chitinase signal peptide, or fragments orvariants thereof, can likewise by assayed using a variety of techniqueswell known to one of ordinary skill in the art, including those thatdetect the ability of the chitinase signal peptide, or fragment orvariant thereof, to direct extracellular secretion of a polypeptide ofinterest.

Methods for mutagenesis and nucleotide sequence alterations are wellknown in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci.USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382;U.S. Pat. No. 4,873,192 (herein incorporated by reference); Walker andGaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, NY) and the references cited therein.

The Lemnaceae expression control elements of the present invention, andvariants or fragments thereof, when assembled within a nucleotideconstruct such that the expression control element is operably linked toa nucleotide sequence of interest, enable expression of the operablylinked nucleotide sequence in a plant or plant cell or nodule (e.g., aduckweed plant or duckweed plant cell or nodule, such as from the genusSpirodela, genus Wolffia, genus Wolfiella, genus Landoltia, or genusLemna). By “operably linked” is intended that the transcription ortranslation of the nucleotide sequence of interest is under theinfluence of the expression control element. In this manner, thenucleotide sequences for the expression control elements of theinvention are provided in expression cassettes or vectors along with thenucleotide sequence of interest, typically a heterologous nucleotidesequence, for expression in the plant or plant cell or nodule. By“heterologous nucleotide sequence” is intended a sequence that is notnaturally operably linked with the expression control element. Whilethis nucleotide sequence is heterologous to the expression controlelement, it may be homologous, or native, or heterologous, or foreign,to the plant host.

It is recognized that the expression control elements of the invention,or variants or fragments thereof, can be used to drive expression of therespective native coding sequence. Such constructs can change expressionlevels of the native polypeptide in the plant or plant cell. Thus, thephenotype of the plant or plant cell can be altered.

As used herein, “vector” refers to a DNA molecule such as a plasmid,cosmid, or bacterial phage for introducing a nucleotide construct, forexample, an expression cassette, into a host cell. Cloning vectorstypically contain one or a small number of restriction endonucleaserecognition sites at which foreign DNA sequences can be inserted in adeterminable fashion without loss of essential biological function ofthe vector, as well as a marker gene, as described herein below, that issuitable for use in the identification and selection of cellstransformed with the cloning vector.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, andprogeny of same. Parts of transgenic plants are to be understood withinthe scope of the invention to comprise, for example, plant cells,protoplasts, tissues, callus, embryos as well as flowers, ovules, stems,fruits, leaves, roots, root tips, and the like originating in transgenicplants or their progeny previously transformed with a DNA molecule ofthe invention and therefore consisting at least in part of transgeniccells. As used herein, the term “plant cell” includes, withoutlimitation, cells of seeds, embryos, meristematic regions, callustissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores. The class of plants that can be used in the methods of theinvention is generally as broad as the class of higher plants amenableto transformation techniques, including both monocotyledonous anddicotyledonous plants. Such plants include, for example, duckweed.

The term “duckweed” refers to members of the family Lemnaceae. Thisfamily currently is divided into five genera and 38 species of duckweedas follows: genus Lemna (L. aequinoctialis, L. disperma, L.ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L.obscura, L. perpusilla, L. tenera, L. trisulca, L. turionifera, L.valdiviana); genus Spirodela (S. intermedia, S. polyrrhiza, S.punctata); genus Wolffia (Wa. angusta, Wa. arrhiza, Wa. australina, Wa.borealis, Wa. brasiliensis, Wa. columbiana, Wa. elongata, Wa. globosa,Wa. microscopica, Wa. neglecta); genus Wolfiella (Wl. caudata, Wl.denticulata, Wl. gladiata, Wl. hyalina, Wl. lingulata, Wl. repunda, Wl.rotunda, and Wl. neotropica) and genus Landoltia (L. punctata). Anyother genera or species of Lemnaceae, if they exist, are also aspects ofthe present invention. Lemna species can be classified using thetaxonomic scheme described by Landolt (1986) Biosystematic Investigationon the Family of Duckweeds: The family of Lemnaceae—A Monograph Study(Geobatanischen Institut ETH, Stiftung Rubel, Zurich).

The term “duckweed nodule” as used herein refers to duckweed tissuecomprising duckweed cells where at least about 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% of the cells are differentiated cells.A “differentiated cell,” as used herein, is a cell with at least onephenotypic characteristic (e.g., a distinctive cell morphology or theexpression of a marker nucleic acid or protein) that distinguishes itfrom undifferentiated cells or from cells found in other tissue types.The differentiated cells of the duckweed nodule culture described hereinform a tiled smooth surface of interconnected cells fused at theiradjacent cell walls, with nodules that have begun to organize into frondprimordium scattered throughout the tissue. The surface of the tissue ofthe nodule culture has epidermal cells connected to each other viaplasmadesmata.

In some embodiments, expression cassettes or vectors comprising aLemnaceae expression control element, or a variant or fragment thereof,operably linked to a nucleotide sequence of interest are provided forexpression of the polypeptide encoded by the nucleotide sequence ofinterest in a plant or plant cell or nodule. The operably linkednucleotide sequence of interest may be any sequence whose expression ina plant or plant cell or nodule is desirable. The nucleotide sequence ofinterest will typically be a heterologous nucleotide sequence, asdefined herein. Exemplary heterologous nucleotide sequences of interestinclude, but are not limited to, sequences that encode mammalianpolypeptides, such as insulin, growth hormone, α-interferon,β-interferon, β-glucocerebrosidase, β-glucoronidase, retinoblastomaprotein, p53 protein, angiostatin, leptin, erythropoietin, granulocytemacrophage colony stimulating factor, plasminogen, monoclonalantibodies, Fab fragments, single chain antibodies, cytokines,receptors, human vaccines, animal vaccines, peptides, and serum albumin.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. As used herein, the terms “encoding” or “encoded” when used inthe context of a specified nucleic acid mean that the nucleic acidcomprises the requisite information to direct translation of thenucleotide sequence into a specified protein. The information by which aprotein is encoded is specified by the use of codons. A nucleic acidencoding a protein may comprise non-translated sequences (e.g., introns)within translated regions of the nucleic acid or may lack suchintervening non-translated sequences (e.g., as in cDNA).

In a specific, non-limiting example, transformed duckweed is obtained bytransformation with an expression cassette comprising a Lemnaceaeubiquitin expression control element (e.g., as set forth in SEQ IDNOs:1-3), a fragment thereof (e.g., as set forth in SEQ ID NOs:4-9), aLemnaceae r-histone expression control element (e.g., as set forth inSEQ ID NO:13), a Lemnaceae chitinase expression control element (e.g.,as set forth in SEQ ID NO:14), or a variant of these sequences operablylinked to a heterologous nucleotide sequence of interest. Transformedduckweed can also be obtained by transformation with an expressioncassette comprising a Lemna gibba RbcS expression control element (e.g.,as set forth in SEQ ID NOs:10-12; see GenBank Accession Nos. S45165(SSU13; nucleotides 694-757), S45166 (SSU5A; nucleotides 698-755) andS45167 (SSU5B; nucleotides 690-751)), or a variant or fragment thereof,operably linked to a heterologous nucleotide sequence of interest. Theexpression control elements set forth in SEQ ID NOs:10-12 advantageouslyenhance expression of operably linked heterologous nucleotide sequencesin transformed duckweed compared to expression without the elements.

An expression cassette of the invention is provided with a plurality ofrestriction sites for insertion of the nucleotide sequence encoding theprotein of interest to be under the transcriptional regulation of theexpression control element. The expression cassette may encode a singlegene of interest. Alternatively, the expression cassette may encode twoor more genes of interest.

The expression cassettes described herein include in the 5′-3′ directionof transcription, a transcriptional and translational initiation region(e.g., an expression control element of the invention or biologicallyactive variant or fragment thereof), a nucleotide sequence of interest,and a transcriptional and translational termination region functional inplants. Any suitable termination sequence known in the art may be usedin accordance with the present invention. The termination region may benative with the transcriptional initiation region, may be native withthe nucleotide sequence of interest, or may be derived from anothersource. Convenient termination regions are available from the Ti-plasmidof A. tumefaciens, such as the octopine synthetase and nopalinesynthetase termination regions. See also Guerineau et al. (1991) Mol.Gen. Genet. 262:141; Proudfoot (1991) Cell 64:671; Sanfacon et al.(1991) Genes Dev. 5:141; Mogen et al. (1990) Plant Cell 2:1261; Munroeet al. (1990) Gene 91:151; Ballas et al. (1989) Nucleic Acids Res.17:7891; and Joshi et al. (1987) Nucleic Acids Res. 15:9627. Additionalexemplary termination sequences are the pea RubP carboxylase smallsubunit termination sequence, the Cauliflower Mosaic Virus 35Stermination sequence, and the ubiquitin terminator from many plantspecies. Other suitable termination sequences will be apparent to thoseskilled in the art.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells or tissues. Selectablemarker genes include genes encoding antibiotic resistance, such as thoseencoding neomycin phosphotransferase II (NEO), neomycinphosphotransferase III and hygromycin phosphotransferase (HPT), as wellas genes conferring resistance to herbicidal compounds. Herbicideresistance genes generally code for a modified target proteininsensitive to the herbicide or for an enzyme that degrades ordetoxifies the herbicide in the plant before it can act. See, DeBlock etal. (1987) EMBO J. 6:2513; DeBlock et al. (1989) Plant Physiol. 91:691;Fromm et al. (1990) BioTechnology 8:833; Gordon-Kamm et al. (1990) PlantCell 2:603; and Frisch et al. (1995) Plant Mol. Biol. 27:405-9. Forexample, resistance to glyphosate or sulfonylurea herbicides has beenobtained using genes coding for the mutant target enzymes,5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and acetolactatesynthase (ALS). Resistance to glufosinate ammonium, boromoxynil, and2,4-dichlorophenoxyacetate (2,4-D) have been obtained by using bacterialgenes encoding phosphinothricin acetyltransferase, a nitrilase, or a2,4-dichlorophenoxyacetate monooxygenase, which detoxify the respectiveherbicides.

For purposes of the present invention, selectable marker genes include,but are not limited to, genes encoding neomycin phosphotransferase II(Fraley et al. (1986) CRC Critical Reviews in Plant Science 4:1),neomycin phosphotransferase III (Frisch et al. (1995) Plant Mol. Biol.27:405-9), cyanamide hydratase (Maier-Greiner et al. (1991) Proc. Natl.Acad. Sci. USA 88:4250); aspartate kinase; dihydrodipicolinate synthase(Perl et al. (1993) BioTechnology 11:715); bar gene (Toki et al. (1992)Plant Physiol. 100: 1503; Meagher et al. (1996) Crop Sci. 36:1367);tryptophan decarboxylase (Goddijn et al. (1993) Plant Mol. Biol.22:907); neomycin phosphotransferase (NEO; Southern et al. (1982) J.Mol. Appl. Gen. 1:327); hygromycin phosphotransferase (HPT or HYG;Shimizu et al. (1986) Mol. Cell. Biol. 6:1074); dihydrofolate reductase(DHFR; Kwok et al. (1986) Proc. Natl. Acad. Sci. USA 83:4552);phosphinothricin acetyltransferase (DeBlock et al. (1987) EMBO J.6:2513); 2,2-dichloropropionic acid dehalogenase (Buchanan-Wollatron etal. (1989) J. Cell. Biochem. 13D:330); acetohydroxyacid synthase (U.S.Pat. No. 4,761,373 to Anderson et al.; Haughn et al. (1988) Mol. Gen.Genet. 221:266); 5-enolpyruvyl-shikimate-phosphate synthase (aroA; Comaiet al. (1985) Nature 317:741); haloarylnitrilase (WO 87/04181 to Stalkeret al.); acetyl-coenzyme A carboxylase (Parker et al. (1990) PlantPhysiol. 92:1220); dihydropteroate synthase (sulI; Guerineau et al.(1990) Plant Mol. Biol. 15:127); and 32 kDa photosystem II polypeptide(psbA; Hirschberg et al. (1983) Science 222:1346 (1983).

Also included are genes encoding resistance to gentamicin (e.g., aacC1,Wohlleben et al. (1989) Mol. Gen. Genet. 217:202-208); chloramphenicol(Herrera-Estrella et al. (1983) EMBO J. 2:987); methotrexate(Herrera-Estrella et al. (1983) Nature 303:209; Meijer et al. (1991)Plant Mol. Biol. 16:807); hygromycin (Waldron et al. (1985) Plant Mol.Biol. 5:103; Zhijian et al. (1995) Plant Science 108:219; Meijer et al.(1991) Plant Mol. Bio. 16:807); streptomycin (Jones et al. (1987) Mol.Gen. Genet. 210:86); spectinomycin (Bretagne-Sagnard et al. (1996)Transgenic Res. 5:131); bleomycin (Hille et al. (1986) Plant Mol. Biol.7:171); sulfonamide (Guerineau et al. (1990) Plant Mol. Bio. 15:127);bromoxynil (Stalker et al. (1988) Science 242:419); 2,4-D (Streber etal. (1989) BioTechnology 7:811); phosphinothricin (DeBlock et al. (1987)EMBO J. 6:2513); spectinomycin (Bretagne-Sagnard and Chupeau, TransgenicResearch 5:131).

The bar gene confers herbicide resistance to glufosinate-typeherbicides, such as phosphinothricin (PPT) or bialaphos, and the like.Other selectable markers that could be used in the expression constructsinclude, but are not limited to, the PAT gene, also for bialaphos andphosphinothricin resistance, the ALS gene for imidazolinone resistance,the HPH or HYG gene for hygromycin resistance, the EPSP synthase genefor glyphosate resistance, the Hm1 gene for resistance to the Hc-toxin,and other selective agents used routinely and known to one of ordinaryskill in the art. See Yarranton (1992) Curr. Opin. Biotech. 3:506;Chistopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314; Yao etal. (1992) Cell 71:63; Reznikoff (1992) Mol. Microbiol. 6:2419; Barkleyet al. (1980) The Operon 177-220; Hu et al. (1987) Cell 48:555; Brown etal. (1987) Cell 49:603; Figge et al. (1988) Cell 52:713; Deuschle et al.(1989) Proc. Natl. Acad. Sci. USA 86:5400; Fuerst et al. (1989) Proc.Natl. Acad. Sci. USA 86:2549; Deuschle et al. (1990) Science 248:480;Labow et al. (1990) Mol. Cell. Biol. 10:3343; Zambretti et al. (1992)Proc. Natl. Acad. Sci. USA 89:3952; Baim et al. (1991) Proc. Natl. Acad.Sci. USA 88:5072; Wyborski et al. (1991) Nuc. Acids Res. 19:4647;Hillenand-Wissman (1989) Topics in Mol. And Struc. Biol. 10:143;Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:1591;Kleinschnidt et al. (1988) Biochemistry 27:1094; Gatz et al. (1992)Plant J. 2:397; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547;Oliva et al. (1992) Antimicrob. Agents Chemother. 36:913; Hlavka et al.(1985) Handbook of Experimental Pharmacology 78; and Gill et al. (1988)Nature 334:721. Such disclosures are herein incorporated by reference.

The above list of selectable marker genes are not meant to be limiting.Any lethal or non-lethal selectable marker gene can be used in thepresent invention.

In some embodiments, the present invention provides for the modificationof the expressed nucleotide sequence of interest to enhance itsexpression in the plant of interest. Methods are available in the artfor synthesizing nucleotide sequences with plant-preferred codons. See,for example, U.S. Pat. Nos. 5,380,831 and 5,436,391 (both of which areherein incorporated by reference); Perlak et al. (1991) Proc. Natl.Acad. Sci. USA 15:3324; Tannacome et al. (1997) Plant Mol. Biol. 34:485;and Murray et al. (1989) Nucleic Acids. Res. 17:477.

For example, where the plant of interest is duckweed, one suchmodification is the synthesis of the nucleotide sequence of interestusing duckweed-preferred codons. The preferred codons may be determinedfrom the codons of highest frequency in the proteins expressed induckweed. Thus, the frequency of usage of particular a codon in duckweedmay be determined by analyzing codon usage in a group of duckweed codingsequences. A number of duckweed coding sequences are known to those ofskill in the art; see for example, the sequences contained in theGenBank® database, which may be accessed through the website for theNational Center for Biotechnology Information, a division of theNational Library of Medicine, which is located in Bethesda, Md. Tablesshowing the frequency of codon usage based on the sequences contained inthe most recent GenBank® release may be found on the website for theKazusa DNA Research Institute in Chiba, Japan. This database isdescribed in Nakamura et al. (2000) Nucleic Acids Res. 28:292.

It is recognized that genes that have been optimized for expression induckweed and other monocots or dicots can be used in the methods of theinvention. See, e.g., EP 0 359 472, EP 0 385 962, WO 91/16432; Perlak etal. (1991) Proc. Natl. Acad. Sci. USA 88:3324; Iannacome et al. (1997)Plant Mol. Biol. 34:485; Murray et al. (1989) Nucleic Acids Res. 17:477;and the like. It is further recognized that all or any part of the genesequence may be optimized or synthetic. In other words, fully optimizedor partially optimized sequences may also be used. For example, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons may beplant-preferred codons, for example, duckweed-preferred codons. Thus, insome embodiments, the nucleotide sequence encoding the polypeptide ofinterest comprises between 50-100% duckweed-preferred codons or between70-100% duckweed-preferred codons. In one embodiment, between 90 and 96%of the codons are duckweed-preferred codons. The coding sequence of thenucleotide sequence of interest may comprise codons used with afrequency of at least 17% in duckweed. Codon usage in Lemna gibba(Table 1) and Lemna minor (Table 2) is shown below. In some embodiments,Table 1 or Table 2 is used to select duckweed-preferred codons.

TABLE 1 Lemna gibba codon usage from GenBank ® Release 139* Amino AcidCodon Number /1000 Fraction Gly GGG 57.00 28.89 0.35 Gly GGA 8.00 4.050.05 Gly GGT 3.00 1.52 0.02 Gly GGC 93.00 47.14 0.58 Glu GAG 123.0062.34 0.95 Glu GAA 6.00 3.04 0.05 Asp GAT 6.00 3.04 0.08 Asp GAC 72.0036.49 0.92 Val GTG 62.00 31.42 0.47 Val GTA 0.00 0.00 0.00 Val GTT 18.009.12 0.14 Val GTC 51.00 25.85 0.39 Ala GCG 44.00 22.30 0.21 Ala GCA14.00 7.10 0.07 Ala GCT 14.00 7.10 0.07 Ala GCC 139.00 70.45 0.66 ArgAGG 16.00 8.11 0.15 Arg AGA 11.00 5.58 0.10 Ser AGT 1.00 0.51 0.01 SerAGC 44.00 22.30 0.31 Lys AAG 116.00 58.79 1.00 Lys AAA 0.00 0.00 0.00Asn AAT 2.00 1.01 0.03 Asn AAC 70.00 35.48 0.97 Met ATG 67.00 33.96 1.00Ile ATA 4.00 2.03 0.06 Ile ATT 0.00 0.00 0.00 Ile ATC 63.00 31.93 0.94Thr ACG 19.00 9.63 0.25 Thr ACA 1.00 0.51 0.01 Thr ACT 6.00 3.04 0.08Thr ACC 50.00 25.34 0.66 Trp TGG 45.00 22.81 1.00 End TGA 4.00 2.03 0.36Cys TGT 0.00 0.00 0.00 Cys TGC 34.00 17.23 1.00 End TAG 0.00 0.00 0.00End TAA 7.00 3.55 0.64 Tyr TAT 4.00 2.03 0.05 Tyr TAC 76.00 38.52 0.95Leu TTG 5.00 2.53 0.04 Leu TTA 0.00 0.00 0.00 Phe TTT 4.00 2.03 0.04 PheTTC 92.00 46.63 0.96 Ser TCG 34.00 17.23 0.24 Ser TCA 2.00 1.01 0.01 SerTCT 1.00 0.51 0.01 Ser TCC 59.00 29.90 0.42 Arg CGG 23.00 11.66 0.22 ArgCGA 3.00 1.52 0.03 Arg CGT 2.00 1.01 0.02 Arg CGC 50.00 25.34 0.48 GlnCAG 59.00 29.90 0.86 Gln CAA 10.00 5.07 0.14 His CAT 5.00 2.53 0.26 HisCAC 14.00 7.10 0.74 Leu CTG 43.00 21.79 0.35 Leu CTA 2.00 1.01 0.02 LeuCTT 1.00 0.51 0.01 Leu CTC 71.00 35.99 0.58 Pro CCG 44.00 22.30 0.31 ProCCA 6.00 3.04 0.04 Pro CCT 13.00 6.59 0.09 Pro CCC 80.00 40.55 0.56

TABLE 2 Lemna minor codon usage from GenBank ® Release 139* AmAcid CodonNumber /1000 Fraction Gly GGG 8.00 17.39 0.22 Gly GGA 11.00 23.91 0.31Gly GGT 1.00 2.17 0.03 Gly GGC 16.00 34.78 0.44 Glu GAG 25.00 54.35 0.78Glu GAA 7.00 15.22 0.22 Asp GAT 8.00 17.39 0.33 Asp GAC 16.00 34.78 0.67Val GTG 21.00 45.65 0.53 Val GTA 3.00 6.52 0.07 Val GTT 6.00 13.04 0.15Val GTC 10.00 21.74 0.25 Ala GCG 13.00 28.26 0.32 Ala GCA 8.00 17.390.20 Ala GCT 6.00 13.04 0.15 Ala GCC 14.00 30.43 0.34 Arg AGG 9.00 19.570.24 Arg AGA 11.00 23.91 0.30 Ser AGT 2.00 4.35 0.05 Ser AGC 11.00 23.910.26 Lys AAG 13.00 28.26 0.68 Lys AAA 6.00 13.04 0.32 Asn AAT 0.00 0.000.00 Asn AAC 12.00 26.09 1.00 Met ATG 9.00 19.57 1.00 Ile ATA 1.00 2.170.08 Ile ATT 2.00 4.35 0.15 Ile ATC 10.00 21.74 0.77 Thr ACG 5.00 10.870.28 Thr ACA 2.00 4.35 0.11 Thr ACT 2.00 4.35 0.11 Thr ACC 9.00 19.570.50 Trp TGG 8.00 17.39 1.00 End TGA 1.00 2.17 1.00 Cys TGT 1.00 2.170.12 Cys TGC 7.00 15.22 0.88 End TAG 0.00 0.00 0.00 End TAA 0.00 0.000.00 Tyr TAT 1.00 2.17 0.12 Tyr TAC 7.00 15.22 0.88 Leu TTG 3.00 6.520.08 Leu TTA 1.00 2.17 0.03 Phe TTT 6.00 13.04 0.25 Phe TTC 18.00 39.130.75 Ser TCG 11.00 23.91 0.26 Ser TCA 4.00 8.70 0.09 Ser TCT 6.00 13.040.14 Ser TCC 9.00 19.57 0.21 Arg CGG 4.00 8.70 0.11 Arg CGA 4.00 8.700.11 Arg CGT 0.00 0.00 0.00 Arg CGC 9.00 19.57 0.24 Gln CAG 11.00 23.910.73 Gln CAA 4.00 8.70 0.27 His CAT 0.00 0.00 0.00 His CAC 6.00 13.041.00 Leu CTG 9.00 19.57 0.24 Leu CTA 4.00 8.70 0.11 Leu CTT 4.00 8.700.11 Leu CTC 17.00 36.96 0.45 Pro CCG 8.00 17.39 0.29 Pro CCA 7.00 15.220.25 Pro CCT 5.00 10.87 0.18 Pro CCC 8.00 17.39 0.29

Other modifications can also be made to the nucleotide sequence ofinterest to optimize its expression in a plant. These modificationsinclude, but are not limited to, elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence may be modified to avoid predicted hairpinsecondary mRNA structures.

There are known differences between the optimal translation initiationcontext nucleotide sequences for translation initiation codons inanimals and plants and the composition of these translation initiationcontext nucleotide sequences can influence the efficiency of translationinitiation. See, for example, Lukaszewicz et al. (2000) Plant Science154:89-98; and Joshi et al. (1997) Plant Mol. Biol. 35:993-1001. In someembodiments of the present invention, the translation initiation contextnucleotide sequence for the translation initiation codon of thenucleotide sequence of interest may be modified to enhance expression induckweed. In one embodiment, the nucleotide sequence is modified suchthat the three nucleotides directly upstream of the translationinitiation codon of the nucleotide sequence of interest are “ACC.” In asecond embodiment, these nucleotides are “ACA.”

In addition to the expression control elements described herein forinitiating or enhancing expression of a heterologous nucleotide sequencein a plant, expression of a nucleotide sequence of interest can also beenhanced by the optional use of various regulatory elements. “Regulatoryelement” as used herein, refers to a nucleotide sequence, either DNA orRNA, usually upstream (5′) of the coding sequence of a structural gene,including transcriptional control sequences such as leader sequences,promoters, translational and transcriptional enhancers or repressors,and mRNA stability and instability determinants. Sequences found withinintrons may also regulate expression of the coding region of interest.Regulatory elements can also be found 3′ to the site of transcriptioninitiation, or within transcribed regions. The various regulatoryelements can be operably linked to other regulatory elements. “Leadersequence” as used herein refers to the portion of a nucleic acid locatedat the 5′ end of mRNA, extending from the 5′CAP site to the AUG proteintranslation initiation codon. The leader sequence is important intranslation initiation and in gene expression regulation.

For example, one or more leader sequences may additionally be used incombination to enhance expression of the target nucleotide sequence.Translation leaders are known in the art and include, but are notlimited to, picornavirus leaders, e.g., EMCV leader(Encephalomyocarditis 5′ noncoding region; Elroy-Stein et al. (1989)Proc. Natl. Acad. Sci. USA 86:6126); potyvirus leaders, e.g., TEV leader(Tobacco Etch Virus; Gallie et al. (1995) Gene 165:233); humanimmunoglobulin heavy-chain binding protein (BiP; Macajak and Sarnow(1991) Nature 353:90); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4; Jobling and Gehrke (1987) Nature325:622); tobacco mosaic virus leader (TMV; Gallie (1989) MolecularBiology of RNA, 23:56); potato etch virus leader (Tomashevskaya et al.(1993) J. Gen. Virol. 74:2717-2724); Fed-1 5′ untranslated region(Dickey (1992) EMBO J. 11:2311-2317); RbcS 5′ untranslated region(Silverthome et al. (1990) J. Plant. Mol. Biol. 15:49-58); and maizechlorotic mottle virus leader (MCMV; Lommel et al. (1991) Virology81:382). See also, Della-Cioppa et al. (1987) Plant Physiology 84:965.Leader sequences comprising a plant intron sequence, including theintron sequence from the maize dehydrogenase 1 gene, the castor beancatalase gene, or the Arabidopsis tryptophan pathway gene PAT1, havealso been shown to increase translational efficiency in plants (Calliset al. (1987) Genes Dev. 1:1183-1200; Mascarenhas et al. (1990) PlantMol. Biol. 15:913-920).

The Lemnaceae ubiquitin introns described herein above (i.e., as setforth in SEQ ID NOs:7-9) can be used with promoters other than theirrespective ubiquitin promoters to enhance expression of an operablylinked nucleotide sequence of interest. The promoter used with theubiquitin introns can be any promoter suitable for use in the plant ofinterest, including the novel Lemnaceae r-histone and chitinasepromoters disclosed in SEQ ID NOs:13 and 14, respectively. Othersuitable promoters can be obtained from a variety of sources, such asplants or plant DNA viruses. Useful promoters include those isolatedfrom the caulimovirus group, such as the cauliflower mosaic virus 19Sand 35S (CaMV19S and CaMV35S) transcript promoters. Other usefulpromoters include the enhanced CaMV35S promoter (eCaMV35S) as describedby Kat et al. (1987) Science 236:1299-1302, and the small subunitpromoter of ribulose 1,5-bisphosphate carboxylase oxygenase (RUBISCO).Examples of other suitable promoters are rice actin promoter;cyclophilin promoter; ADH1 promoter (Callis et al. (1987) Gene Dev.1:1183-1200); Class I patatin promoter (Bevan et al. (1986) Nucl. AcidsRes. 14:4675-4638); ADP glucose pyrophosphorylase promoter;β-conglycinin promoter (Tiemey et al. (1987) Planta 172:356-363); E8promoter (Deikman et al. (1988) Embo J. 7:3315-3320); 2AII promoter(Pear et al. (1989) Plant Mol. Biol. 13:639-651); and acid chitinasepromoter (Samac et al. (1990) Plant Physiol. 93:907-914).

It is recognized that any of the expression-enhancing nucleotidesequence modifications described above can be used in the presentinvention, including any single modification or any possible combinationof modifications.

In some embodiments, the compositions and methods of the invention areutilized in a plant expression system, for example, a duckweedexpression system, and the heterologous nucleotide sequence of interestis a secreted protein. Secreted proteins are usually translated fromprecursor polypeptides that include a “signal peptide” that interactswith a receptor protein on the membrane of the endoplasmic reticulum(ER) to direct the translocation of the growing polypeptide chain acrossthe membrane and into the endoplasmic reticulum for secretion from thecell. This signal peptide is often cleaved from the precursorpolypeptide to produce a “mature” polypeptide lacking the signalpeptide. In this manner, a biologically active polypeptide is expressedin a plant, for example, duckweed, from an expression construct havingan expression control element of the invention, or a biologically activevariant or fragment thereof, operably linked to a nucleotide sequence ofinterest that is further operably linked with a nucleotide sequenceencoding a signal peptide that directs secretion of the polypeptide intothe culture medium. A “biologically active polypeptide” refers to apolypeptide that has the capability of performing one or more biologicalfunctions or a set of activities normally attributed to the polypeptidein a biological context. Plant signal peptides that target proteintranslocation to the endoplasmic reticulum (for secretion outside of thecell) are known in the art. See, for example, U.S. Pat. No. 6,020,169,herein incorporated by reference.

In one embodiment, the signal peptide is the novel L. minor chitinasesignal peptide set forth in SEQ ID NO:16, or a variant or fragmentthereof, and the expression construct includes a nucleotide sequenceencoding this signal peptide operably linked to a nucleotide sequence ofinterest. In some embodiments, this signal peptide-encoding sequence isthe sequence set forth in SEQ ID NO:15.

It is recognized that the L. minor chitinase signal peptide of theinvention, or variants or fragments thereof, can be used to directextracellular secretion of any encoded polypeptide of interest. In thismanner, the signal peptide-encoding sequence of SEQ ID NO:15, or avariant or fragment thereof, can be incorporated into any expressionconstruct such that it is operably linked in proper reading frame to apromoter of interest and a polypeptide-encoding nucleotide sequence ofinterest. Such an expression construct can be introduced into a plant orplant cell or nodule to provide for expression and extracellularsecretion of the encoded polypeptide of interest.

Alternatively, a mammalian signal peptide can be used to targetrecombinant polypeptides expressed in genetically engineered plants, forexample, duckweed, for secretion. It has been demonstrated that plantcells recognize mammalian signal peptides that target the endoplasmicreticulum, and that these signal peptides can direct the secretion ofpolypeptides not only through the plasma membrane but also through theplant cell wall. See, for example, U.S. Pat. Nos. 5,202,422 and5,639,947, both of which are incorporated herein by reference.

The secreted polypeptide can be harvested from the culture medium by anyconventional means known in the art and purified by chromatography,electrophoresis, dialysis, solvent-solvent extraction, and the like.

The methods of the invention involve introducing an expression constructinto a plant or plant cell or nodule. By “introducing” is intendedpresenting to the plant an expression construct in such a manner thatthe construct gains access to the interior of a cell of the plant. Themethods of the invention do not depend on a particular method forintroducing an expression construct to a plant, only that the expressionconstruct gains access to the interior of at least one cell of theplant. Methods for introducing expression constructs into plants areknown in the art including, but not limited to, stable transformationmethods, transient transformation methods, and virus-mediated methods.

By “stable transformation” is intended that a nucleotide sequenceintroduced into a plant integrates into the genome of the plant and iscapable of being inherited by progeny thereof. By “transienttransformation” is intended that a nucleotide sequence (e.g., anucleotide sequence contained in an expression construct) introducedinto a plant does not integrate into the genome of the plant.

The nucleotide sequences of the invention may be introduced into plantsor plant cells or nodules by contacting the plants or plant cells ornodules with a virus or viral nucleic acids. Generally, such methodsinvolve incorporating a nucleotide sequence of the invention within aviral DNA or RNA molecule. Methods for introducing nucleotide sequencesinto plants or plant cells or nodules and expressing a protein encodedtherein, involving viral DNA or RNA molecules, are known in the art.See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785,5,589,367, and 5,316,931, each of which is herein incorporated byreference.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell or nodule, that is, monocot or dicot, targeted for transformation.Suitable methods of introducing nucleotide sequences into plants orplant cells or nodules include microinjection (Crossway et al. (1986)Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc.Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediatedtransformation (U.S. Pat. Nos. 5,563,055 and 5,981,840, both of whichare herein incorporated by reference), direct gene transfer (Paszkowskiet al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration(see, e.g., U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; and5,932,782 (each of which is herein incorporated by reference); and Tomeset al. (1995) “Direct DNA Transfer into Intact Plant Cells viaMicroprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin);McCabe et al. (1988) Biotechnology 6:923-926). The cells that have beentransformed may be grown into plants in accordance with conventionalways. See, for example, McCormick et al. (1986) Plant Cell Reports5:81-84.

In some embodiments, the stably transformed duckweed plants or duckweedplant cells or nodules express biologically active polypeptides thatcannot effectively be commercially produced by existing gene expressionsystems, because of cost or logistical constraints, or both. Forexample, some proteins cannot be expressed in mammalian systems becausethe protein interferes with cell viability, cell proliferation, cellulardifferentiation, or protein assembly in mammalian cells. Such proteinsinclude, but are not limited to, retinoblastoma protein, p53,angiostatin, and leptin. The present invention can be advantageouslyemployed to produce mammalian regulatory proteins; it is unlikely giventhe large evolutionary distance between higher plants and mammals thatthese proteins will interfere with regulatory processes in duckweed.Transgenic duckweed can also be used to produce large quantities ofproteins such as serum albumin (in particular, human serum albumin),hemoglobin, and collagen, which challenge the production capabilities ofexisting expression systems.

Additionally, higher plant systems can be engineered to producebiologically active multimeric proteins (e.g., monoclonal antibodies,hemoglobin, P450 oxidase, and collagen, and the like) far more easilythan can mammalian systems. One exemplary approach for producingbiologically active multimeric proteins in duckweed uses an expressionconstruct containing the genes encoding all of the polypeptide subunits.See, e.g., During et al. (1990) Plant Mol. Biol. 15:281 and van Engelenet al. (1994) Plant Mol. Biol. 26:1701. This construct is thenintroduced into duckweed cells using any known transformation method,such as a ballistic bombardment or Agrobacterium-mediatedtransformation. This method results in clonal cell lines that expressall of the polypeptides necessary to assemble the multimeric protein. Avariation on this approach is to make single gene constructs, mix DNAfrom these constructs together, then deliver this mixture of DNAs intoplant cells using ballistic bombardment or Agrobacterium-mediatedtransformation. As a further variation, some or all of the constructsmay encode more than one subunit of the multimeric protein (i.e., sothat there are fewer duckweed clones to be crossed than the number ofsubunits in the multimeric protein). Alternatively, each duckweed cloneexpresses at least one of the subunits of the multimeric protein, andduckweed clones secreting each subunit are cultured together and themultimeric protein is assembled in the media from the various secretedsubunits. In some instances, it may be desirable to produce less thanall of the subunits of a multimeric protein, or even a single proteinsubunit, in a transformed duckweed plant or duckweed nodule culture, forexample, for industrial or chemical processes or for diagnostic,therapeutic, or vaccination purposes.

The following examples are offered for purposes of illustration, not byway of limitation.

EXPERIMENTAL Example 1 Expression Vectors

The expression vectors used in the examples described below includeEgs05, Egs07, Egs11, Egs22, Egs23, Egs46, Egs50, Egs51, Egs19, Egs20,Egs24, Egs25, IFN53, and IFN54. Egs05 and Egs07 are unmodifiedexpression vectors comprising a control promoter operably linked to thecoding sequence for E. coli β-glucuronidase (GUS), each with a differentselectable marker gene. Egs11 comprises the full-length L. minorubiquitin expression control element (SEQ ID NO:1) operably linked tothe GUS coding sequence, with a selectable marker gene. Egs22 and Egs23are similar constructs, but use truncated versions of the L. minorubiquitin expression control element. In Egs22, nucleotides 1288-2160 ofSEQ ID NO:1 drive expression of the operably linked GUS coding sequence.In Egs23, nucleotides 1132-2160 of SEQ ID NO:1 drive expression of thisGUS coding sequence. Egs46 is similar to Egs11, but comprises adifferent selectable marker gene.

Egs50 comprises the full-length S. polyrrhiza ubiquitin expressioncontrol element (SEQ ID NO:2) operably linked to the GUS codingsequence, with a selectable marker gene. Similarly, Egs51 comprises thefull-length L. aequinoctialis ubiquitin expression control element (SEQID NO:3) operably linked to the GUS coding sequence, with a selectablemarker gene.

Egs19 comprises nucleotides 461-1808 of the L. minor r-histoneexpression control element (SEQ ID NO:13) operably linked to the GUScoding sequence, with a selectable marker gene. In Egs20, nucleotides805-1808 of SEQ ID NO:13 drive expression of the GUS coding sequence.

Egs24 comprises nucleotides 51-1338 of the L. minor chitinase expressioncontrol element (SEQ ID NO:14) operably linked to the GUS codingsequence, with a selectable marker gene. Egs25 comprises nucleotides51-1338 of the L. minor chitinase expression control element (SEQ IDNO:14) operably linked to the maize ADH1 intron and GUS coding sequence,with a selectable marker gene.

The IFN53 and IFN54 expression vectors each contain the AmasPmas superpromoter, L. gibba RbcS SSU5B expression control element (SEQ ID NO:12),and maize ADH1 intron operably linked to a codon-optimized interferonalpha-2b gene, with either a codon-optimized alpha amylase signalsequence (IFN53) or the L. minor chitinase signal sequence (SEQ IDNO:15; IFN54).

Example 2 Transformation of Duckweed

Duckweed fronds or duckweed nodule cultures (derived from Lemna minorstrain 8627 in these examples) were transformed with the expressionconstructs described above using Agrobacterium-mediated transformationmethods. Agrobacterium tumefaciens strain C58Z707, a disarmed, broadhost range C58 strain (Hepburn et al. (1985) J. Gen. Microbiol.131:2961-2969) is used for transformation in these examples. Theexpression constructs described above were mobilized into A. tumefaciensby electroporation, or by a triparental mating procedure using E. coliMM294 harboring the mobilizing plasmid pRK2013 (Hoekema et al. (1983)Nature 303:179-180; Ditta et al. (1980) Proc. Natl. Acad. Sci. USA77:7347-7350). C58Z707 strains comprising the expression constructsdescribed above are streaked on AB minimal medium (Chilton et al. (1974)Proc. Nat. Acad. Sci. USA 71:3672-3676) or in YEB or LB medium (1 g/Lyeast extract, 5 g/L beef extract, 5 g/L peptone, 5 g/L sucrose, 0.5 g/LMgSO₄) containing streptomycin at 500 mg/L, spectinomycin at 50 mg/L andkanamycin sulfate at 50 mg/L and grown overnight at 28° C.

Duckweed nodule cultures for transformation were produced as follows.Duckweed fronds were separated, the roots are cut off with a sterilescalpel, and the fronds are placed, ventral side down, on Murashige andSkoog medium (catalog number M-5519; Sigma Chemical Corporation, St.Louis, Mo.) pH 5.6, supplemented with 5 μM 2,4-dichlorophenoxyaceticacid, 0.5 μM 1-Phenyl-3(1,2,3-thiadiazol-5-yl) urea thidiazuron (SigmaP6186), 3% sucrose, 0.4 Difco Bacto-agar (Fisher Scientific), and 0.15%Gelrite (Sigma). Fronds were grown for 5-6 weeks. At this time, thenodules (small, yellowish cell masses) appeared, generally from thecentral part of the ventral side. This nodule tissue was detached fromthe mother frond and cultured in Murashige and Skoog medium supplementedwith 3% sucrose, 0.4% Difco Bacto-agar, 0.15% Gelrite, 1 μM2,4-dichlorophenoxyacetic acid, and 2 μM benzyladenine.

Duckweed nodule cultures were transformed as follows. The appropriateAgrobacterium tumefaciens strain was grown on potato dextrose agar orYEB or LB agar with 50 mg/L kanamycin and 100 μM acetosyringone, andresuspended in Murashige and Skoog medium supplemented with 0.6 MMannitol and 100 μM acetosyringone. Nodule culture tissue was inoculatedby immersing in the solution of resuspended bacteria for 1-2 minutes,blotted to remove excess fluid, and plated on co-cultivation mediumconsisting of Murashige and Skoog medium supplemented with auxin andcytokinin optimized to promote nodule growth and 100 μM acetosyringone.See, Yamamoto et al. (2001) In Vitro Cell Dev. Biol. Plant 37:349-353.

For selection, nodule culture tissue was transferred to regenerationmedium; 0.5× Schenk and Hildebrandt medium supplemented with 1% sucrose0.4% Difco Bacto-Agar, 0.15% Gelrite 500 mg/L cefotaxime, and 6 mg/Lgeneticin and cultured for approximately 6-8 weeks under continuouslight (20-40 μM/m²·sec). The nodule tissue was transferred every 7 daysto fresh culture medium. Selection is complete when the nodule tissueshows vigorous growth on the selection agent.

Example 3 Transient Expression of E. coli GUS in Duckweed

Transient GUS expression was assessed in duckweed nodule culturestransformed with the Egs05, Egs07, Egs11, Egs22, Egs23, Egs46, Egs50,Egs51, Egs19, Egs20, Egs24, and Egs25 constructs. All constructs werecapable of driving strong expression of GUS, as determined by 24 hourstaining (Table 3).

Additionally, GUS enzyme assays were carried out on duckweed nodulecultures transformed with the Egs07, Egs46, Egs50, and Egs51 constructs.The 36 Egs07 transgenic lines averaged 1.345% GUS, the 29 Egs46transgenic lines averaged 2.320% GUS, the 4 Egs50 transgenic linesaveraged 4.008% GUS, and the 8 Egs51 transgenic lines averaged 6.682%GUS.

TABLE 3 Transient GUS Expression in Callus Test Vector Promoter StainingEgs05 control ++++ Egs07 control ++++ Egs11 LmUBQ ++++ Egs22 LmUBQ(trunc #1) +++ Egs23 LmUBQ (trunc #2) ++++ Egs46 LmUBQ ++++ Egs50 SpUBQ++++ Egs51 LaUBQ +++ Egs19 LmHIS (461-1808) ++ Egs20 LmHIS (805-1808) +Egs24 LmCHT (51-1338) + Egs25 LmCHT (51-1338) + ADH1 intron ++ 24 hourstaining; rated on a scale of 1 to 4.

Example 4 Expression of Interferon in Duckweed

Several hundred transgenic duckweed lines were produced using the IFN53and IFN54 constructs and subsequently screened for interferon expressionby ELISA. Similar levels of interferon expression were observed for thetwo constructs. IFN53: top expresser, 1735.66 ng/ml; mean expression,362.04 ng/ml. IFN54: top expresser, 1173.81 ng/ml; mean expression,347.40 ng/ml.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. An isolated nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of: a) a nucleotide sequencecomprising the sequence set forth in SEQ ID NO: 2; b) a nucleotidesequence comprising at least 350 contiguous nucleotides of the sequenceset forth in SEQ ID NO: 2 or 5, wherein said nucleotide sequenceinitiates transcription in a plant cell, and wherein the nucleotidesequence comprises a TATA box; and c) a nucleotide sequence comprising afunctional fragment having at least 15 contiguous nucleotides of thesequence set forth in SEQ ID NO: 2 or 5, wherein said fragment initiatestranscription in a plant cell.
 2. An expression construct comprising thenucleic acid molecule of claim
 1. 3. The expression construct of claim2, further comprising an operably linked heterologous nucleotidesequence of interest.
 4. The expression construct of claim 3, furthercomprising an operably linked coding sequence for a signal peptide thatdirects secretion of a polypeptide encoded by said heterologousnucleotide sequence of interest into culture medium.
 5. The expressionconstruct of claim 4, wherein said signal peptide comprises an aminoacid sequence selected from the group consisting of: a) the sequence setforth in SEQ ID NO:16; b) a sequence having at least 95% sequenceidentity to the sequence set forth in SEQ ID NO:16, wherein saidsequence directs secretion of said polypeptide into culture medium; andc) a functional fragment of the sequence set forth in SEQ ID NO:16,wherein said fragment directs secretion of said polypeptide into culturemedium.
 6. The expression construct of claim 5, wherein said codingsequence for said signal peptide comprises a nucleotide sequenceselected from the group consisting of: a) the sequence set forth in SEQID NO:15; b) a sequence having at least 95% sequence identity to thesequence set forth in SEQ ID NO:15; and c) a fragment of the sequenceset forth in SEQ ID NO:15.
 7. The expression construct of claim 3,wherein said heterologous nucleotide sequence of interest encodes amammalian polypeptide.
 8. The expression construct of claim 7, whereinsaid mammalian polypeptide is selected from the group consisting ofinsulin, growth hormone, α-interferon, β-interferon,β-glucocerebrosidase, β-glucoronidase, retinoblastoma protein, p53protein, angiostatin, leptin, erythropoietin, granulocyte macrophagecolony stimulating factor, plasminogen, monoclonal antibodies, Fabfragments, single chain antibodies, cytokines, receptors, humanvaccines, animal vaccines, peptides, serum albumin, and combinationsthereof.
 9. A plant or plant cell or nodule transformed with theexpression construct of claim
 2. 10. The transformed plant or plant cellor nodule of claim 9, wherein said plant or plant cell or nodule is amonocot.
 11. The transformed plant or plant cell or nodule of claim 10,wherein said monocot is from a genus selected from the group consistingof the genus Spirodela, genus Wolffia, genus Wolfiella, genus Landoltia,and genus Lemna.
 12. The transformed plant or plant cell or nodule ofclaim 10, wherein said monocot is a member of a species selected fromthe group consisting of Lemna minor, Lemna miniscula, Lemnaaequinoctialis, and Lemna gibba.
 13. The transformed plant or plant cellor nodule of claim 9, wherein said plant or plant cell or nodule is adicot.
 14. An isolated nucleic acid molecule comprising a nucleotidesequence selected from the group consisting of: a) a nucleotide sequencecomprising the sequence set forth in SEQ ID NO:8; and b) a nucleotidesequence comprising a functional fragment of the sequence set forth inSEQ ID NO: 8, wherein said fragment enhances transcription in a plantcell.
 15. An expression construct comprising the nucleic acid moleculeof claim
 14. 16. The expression construct of claim 15, furthercomprising an operably linked promoter of interest.
 17. The expressionconstruct of claim 16, wherein said promoter of interest comprises thenucleotide sequence set forth in SEQ ID NO:5.
 18. A method forexpressing a nucleotide sequence in a plant or plant cell or nodule,said method comprising introducing into the plant or plant cell ornodule an expression construct, said expression construct comprising anexpression control element operably linked to a heterologous nucleotidesequence of interest, wherein said expression control element comprisesa nucleotide sequence selected from the group consisting of: a) anucleotide sequence comprising the sequence set forth in SEQ ID NO: 2;b) a nucleotide sequence comprising at least 350 contiguous nucleotidesof the sequence set forth in SEQ ID NO: 2 or 5, wherein said nucleotidesequence initiates transcription in a plant cell, and wherein thenucleotide sequence comprises a TATA box; and c) a nucleotide sequencecomprising a functional fragment having at least 15 contiguousnucleotides of the sequence set forth in SEQ ID NO: 2 or 5, wherein saidfragment initiates transcription in a plant cell.
 19. The method ofclaim 18, wherein said expression construct further comprises anoperably linked coding sequence for a signal peptide that directssecretion of a polypeptide encoded by said heterologous nucleotidesequence of interest into culture medium.
 20. The method of claim 18,wherein said heterologous nucleotide sequence of interest encodes amammalian polypeptide.
 21. The method of claim 18, wherein saidheterologous nucleotide sequence of interest comprises plant-preferredcodons in the coding sequence for said heterologous nucleotide sequenceof interest.
 22. The expression construct of claim 5, wherein saidheterologous nucleotide sequence of interest encodes a mammalianpolypeptide.
 23. The expression construct of claim 22, wherein saidmammalian polypeptide is selected from the group consisting of insulin,growth hormone, α-interferon, β-interferon, β-glucocerebrosidase,β-glucoronidase, retinoblastoma protein, p53 protein, angiostatin,leptin, erythropoietin, granulocyte macrophage colony stimulatingfactor, plasminogen, monoclonal antibodies, Fab fragments, single chainantibodies, cytokines, receptors, human vaccines, animal vaccines,peptides, serum albumin, and combinations thereof.